High Fidelity Restriction Endonucleases

ABSTRACT

Compositions and methods are provided for enzymes with altered properties that involve a systematic approach to mutagenesis and a screening assay that permits selection of the desired proteins. Embodiments of the method are particularly suited for modifying specific properties of restriction endonucleases such as star activity. The compositions includes restriction endonucleases with reduced star activity as defined by an overall fidelity index improvement factor.

CROSS REFERENCE

This application is a divisional of U.S. Ser. No. 12/172,963 filed Jul.14, 2008 which claims priority from U.S. provisional application Ser.No. 60/959,203 filed Jul. 12, 2007, herein incorporated by reference.

BACKGROUND

Restriction endonucleases are enzymes that cleave double-stranded DNAsin a sequence-specific manner (Roberts, R. J., Proc Natl Acad Sci USA,102:5905-5908 (2005); Roberts, et al., Nucleic Acids Res, 31:1805-1812(2003); Roberts, et al., Nucleic Acids Res, 33:D230-232 (2005); Alves,et al., Restriction Endonucleases, “Protein Engineering of RestrictionEnzymes,” ed. Pingoud, Springer-Verlag Berlin Heidelberg, New York,393-407 (2004)). They are ubiquitously present among prokaryoticorganisms (Raleigh, et al., Bacterial Genomes Physical Structure andAnalysis, Ch. 8, eds. De Bruijin, et al., Chapman & Hall, New York,78-92 (1998)), in which they form part of restriction-modificationsystems, which mainly consist of an endonuclease and amethyltransferase. The cognate methyltransferase methylates the samespecific sequence that its paired endonuclease recognizes and rendersthe modified DNA resistant to cleavage by the endonuclease so that thehost DNA can be properly protected. However, when there is an invasionof foreign DNA, in particular bacteriophage DNA, the foreign DNA will bedegraded before it can be completely methylated. The major biologicalfunction of the restriction-modification system is to protect the hostfrom bacteriophage infection (Arber, Science, 205:361-365 (1979)). Otherfunctions have also been suggested, such as involvement in recombinationand transposition (Carlson, et al., Mol Microbiol, 27:671-676 (1998);Heitman, Genet Eng (NY), 15:57-108 (1993); McKane, et al., Genetics,139:35-43 (1995)).

The specificity of the approximately 3,000 known restrictionendonucleases for their greater than 250 different target sequencescould be considered their most interesting characteristic. After thediscovery of the sequence-specific nature of the first restrictionendonuclease (Danna, et al., Proc Natl Acad Sci U S A, 68:2913-2917(1971); Kelly, et al., J Mol Biol, 51:393-409 (1970)), it did not takelong for scientists to find that certain restriction endonucleasescleave sequences which are similar but not identical to their definedrecognition sequences under non-optimal conditions (Polisky, et al.,Proc Natl Acad Sci USA, 72:3310-3314 (1975); Nasri, et al., NucleicAcids Res, 14:811-821 (1986)). This relaxed specificity is referred toas star activity of the restriction endonuclease. It has been suggestedthat water-mediated interactions between the restriction endonucleaseand DNA are the key differences between specific complexes and starcomplexes (Robinson, et al., J Mol Biol, 234:302-306 (1993); Robinson,et al., Proc Natl Acad Sci USA, 92:3444-3448 (1995), Sidorova, et al.,Biophys J, 87:2564-2576 (2004)).

Star activity is a problem in molecular biology reactions. Star activityintroduces undesirable cuts in a cloning vector or other DNA. In casessuch as forensic applications, where a certain DNA substrate needs to becleaved by a restriction endonuclease to generate a unique fingerprint,star activity will alter a cleavage pattern profile, therebycomplicating analysis. Avoiding star activity is also critical inapplications such as strand displacement amplification (Walker, et al.,Proc Natl Acad Sci USA, 89:392-396 (1992)) and serial analysis of geneexpression (Velculescu, et al., Science, 270:484-487 (1995)).

SUMMARY

In an embodiment of the invention, a composition is provided thatincludes a restriction endonuclease having at least one artificiallyintroduced mutation and an overall fidelity index (FI) improvementfactor of at least two, the restriction endonuclease being capable ofcleaving a substrate with at least a similar cleavage activity to thatof the restriction endonuclease absent the artificially introducedmutation in a predetermined buffer, the artificially introduced mutationbeing the product of at least one of a targeted mutation, saturationmutagenesis, or a mutation introduced through a PCR amplificationprocedure.

In a further embodiment of the invention, at least one of theartificially introduced mutations is a targeted mutation resulting fromreplacement of a naturally occurring residue with an oppositely chargedresidue. An Alanine or a Phenylalanine may replace the naturallyoccurring residue at the target site.

In a further embodiment of the invention, a composition of the typedescribed above includes a restriction enzyme absent the artificiallyintroduced mutation selected from the group consisting of: BamHI, EcoRI,ScaI, SalI, SphI, PstI, NcoI, NheI, SspI, NotI, SacI, PvuII, MfeI,HindIII, SbfI, EagI, EcoRV, AvrII, BstXI, PciI, HpaI, AgeI, BsmBI,BspQI, SapI, KpnI and BsaI.

Further embodiments of the invention include compositions listed inTable 4.

In a further embodiment of the invention, a DNA encoding any of theenzymes listed in Table 4 is provided, a vector comprising the DNA and ahost cell for expressing the protein from the vector.

In an embodiment of the invention, a method is provided having the stepsof (a) identifying which amino acid residues in an amino acid sequenceof a restriction endonuclease having star activity are charged aminoacids; (b) mutating one or more codons encoding one or more of thecharged residues in a gene sequence encoding the restrictionendonuclease; (c) generating a library of gene sequences having one ormore different codon mutations in different charged residues; (d)obtaining a set of proteins expressed by the mutated gene sequences; and(e) determining an FI in a predetermined buffer and a cleavage activityfor each expressed protein.

An embodiment of the method includes the step of determining an overallFI improvement factor for proteins belonging to the set of proteins in adefined set of buffers where for example, the set of buffers containsNEB1, NEB2, NEB3 and NEB4 buffers.

An embodiment of the method includes the steps described above andadditionally mutating codons encoding hydroxylated amino acids or amideamino acids in a same or subsequent step to that of mutating codons forthe charged amino acids.

In an embodiment of the invention described above, the codons aremutated to an Alanine except for Tyrosine which is mutated to aPhenylalanine.

In a further embodiment, the overall FI improvement factor is improvedusing saturation mutagenesis of one or more of the mutated codon.

BRIEF DESCRIPTION OF THE DRAWINGS

In each of the reactions described in FIGS. 1-2A-B, the reaction mixturecontains a volume of 3 μl unless otherwise specified of a buffer fromNew England Biolabs, Inc. (NEB), Ipswich, MA, (see Table 1 and NEBcatalog), 3 μl unless otherwise specified of a specified restrictionendonuclease in a diluent from NEB, Ipswich, Mass. (See Table 1 and NEBcatalog) as well as variable volumes of specified substrate (containing0.6 μg) substrate and a volume of water to bring the reaction mixture toa total of 30 μl. Reactions were conducted at 37° C. for an incubationtime of 1 hour. The results are analyzed on a 0.8% agarose gel. Wherethe overall volume of the reaction mix, amount of substrate, temperatureof the reaction or incubation time varies from above, values areprovided in the description of the figures.

FIG. 1 shows the determination of the FI for wild type (WT) ScaI bydigesting 1.2 μl lambda DNA substrate (0.6 μg) with a two-fold serialdilution using diluent A of a preparation of WT ScaI (1,200 U) in NEB3buffer and examining the digestion products on an agarose gel. Thehighest concentration of a restriction endonuclease with no staractivity is shown with a solid arrow; and the minimum concentrationgiving rise to complete digestion of substrate is shown with a hollowarrow.

FIGS. 2A-B show cleavage of 1.2 μl pXba DNA substrate using 2-foldserial dilutions of NheI-HF (287,200 U and 76.8 U) and 2-fold serialdilutions of WT NheI (9,600 U and 300 U) in NEB3 and NEB4 buffers,respectively. Serial dilutions were performed in diluent A.

FIG. 2A shows cleavage by NheI-HF in NEB4 buffer (upper panel) and NEB3buffer (lower panel).

FIG. 2B shows cleavage by WT NheI in NEB4 buffer (upper panel) and NEB3buffer (lower panel).

The theoretical digestion pattern is provided on the right side of thegel for FIGS. 1-2A-B. Those substrates with only one restrictionendonuclease site should be digested into one linear band fromsupercoiled form.

For FIG. 2A-B “U” denotes units of enzyme.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the invention provide a general method for selecting forrestriction endonucleases with desired characteristics. The generalmethod relies on a suitable assay for determining whether the desiredrestriction endonuclease has been created. In particular an embodimentof the general method provides a systematic screening method with a setof steps. This method has been deduced by performing many hundreds ofreactions using many restriction endonucleases. The example providedherein relates to identifying a restriction endonuclease with reducedstar activity but with cleavage activity that is at least similar to theWT restriction endonuclease. However, it is expected that the samemethodology can be applied successfully to modifying other properties ofthe restriction endonucleases relating, for example, to improvedcleavage activity in desired buffers, thermostability, rate of reactionin defined conditions, etc.

As discussed above, an end point of interest is to transform restrictionendonucleases with star activity into high fidelity restrictionendonucleases with significantly reduced star activity. Star activityrefers to promiscuity in cleavage specificity by individual restrictionendonucleases. The terms “reduction in star activity” and “increase infidelity” are used interchangeably here. Although restrictionendonucleases are characterized by their property of cleaving DNA atspecific sequences, some restriction endonucleases additionally cleaveDNA inefficiently at secondary sites in the DNA. This secondary cleavagemay occur consistently or may arise only under certain conditions suchas any of: increased concentrations, certain buffers, temperature,substrate type, storage, and incubation time.

It is generally acknowledged that little is known about the complexenvironment generated by the hundreds of amino acids that constitute aprotein and determine specificity. One approach in the prior art hasbeen to utilize crystallography to identify contact points between anenzyme and its substrate. Nonetheless, crystallography has limitationswith respect to freezing a structure in time in an unnatural chemicalenvironment.

The rules that determine the contribution of amino acids at any site inthe protein and the role played by the structure of the substratemolecule has proved elusive using existing analytical techniques. Forexample, it is shown here that mutating an amino acid in a restrictionendonuclease can cause all or partial loss of activity.

In this context, no structural explanation has been put forward toexplain why star activity could increase with high glycerolconcentration (>5% v/v), high enzyme to DNA ratio (usually >100 units ofenzyme per μg of DNA), low ionic strength (<25 mM salt), high pH (>8.0),presence of organic solvent (such as DMSO, ethanol), and substitution ofMg²⁺ with other divalent cations (Mn²⁺, Co²⁺). It was here recognizedthat because of the diversity of factors affecting star activity, itwould be necessary to conduct comparisons of WT and mutant star activityunder the same reaction conditions and in the same predetermined bufferand to develop a standard reaction condition in which any high fidelityenzyme must be capable of showing the described characteristics even ifthese characteristics were also observed in other reaction conditions.

Present embodiments of the invention are directed to generating modifiedrestriction endonucleases with specific improved properties, namelyenhanced cleavage fidelity without significant reduction in overallcleavage activity or significant loss of yield from the host cells thatmake the protein. The methods that have been developed here for findingmutants with improved properties have resulted from exhaustiveexperimentation and the properties of the resultant enzymes have beendefined in the context of specified conditions. The methods describedherein may be used for altering the enzymatic properties of anyrestriction endonuclease under predetermined conditions, but are notlimited to the specific defined conditions.

Restriction Steps Used to Generate a High Fidelity RestrictionEndonuclease Endonuclease BamHI Comparison of isoschizomer Targeted 22residues to mutate to Ala. 14 mutants obtained, 3 had improved fidelitySaturation mutagenesis on 2 residues-K30 and E86 Recovered E86P aspreferred mutant with greatest reduced star activity in selectedbuffers. Added mutations to E86P. Second round of mutation (Arg, Lys,His, Asp, Glu, Ser, Thr) to Ala and Tyr to Phe. Selected E167 and Y165for saturation mutagenesis and selected E167T and Y165F. E163A/E167T wasselected as preferred high fidelity mutant (BamHI-HF). EcoRI Comparisonof isoschizomer Targeted 42 charged residues to mutate to Ala. No highfidelity mutants Second round of mutation: Target additional 32 chargedresidues to mutate to Ala: Identified K62A. Saturation mutagenesis onK62A. EcoRI(K62E) was selected as a preferred high fidelity mutant(EcoRI-HF). ScaI Comparison of isoschizomers. Targeted 58 chargedresidues to mutate to Ala. Identify 4 mutants Preferred mutant of 4 is(H193A/S201F). This is selected as a preferred high fidelity mutant(ScaI-HF) SalI Target 86 charged residues and mutate to Ala. SalI(R107A) was preferentially selected as a preferred high fidelity mutant(SalI- HF). SphI Target 71 charged residues and mutate to Ala. SphI(K100A) was preferentially selected as a preferred high fidelity mutant(SphI- HF) PstI Target 92 charged amino acids and mutate to Ala. PstI(D91A) was preferentially selected as a preferred high fidelity mutant(PstI-HF) NcoI Target 66 charged residues and mutate to Ala. NcoI(A2T/R31A) was preferentially selected as a preferred high fidelitymutant (NcoI-HF). NheI Target 92 charged residues and mutate to Ala.NheI (E77A) was (Ex. 1) preferentially selected as a preferred highfidelity mutant (NheI- HF) SspI Target 81 charged residues and mutate toAla. No preferential mutants obtained. Target 95 residues to additionalcharged residues and hydroxylated residues to Ala except Tyr. Tyrmutated to Phe. SspI (Y98F) was preferentially selected as a preferredhigh fidelity mutant (SspI-HF) NotI Target 97 charged residues andmutate to Ala. K150A was preferentially selected as a preferred highfidelity mutant (NotIHF) SacI Target 101 charged residues and mutate toAla. SacI (Q117H/R200A) was preferentially selected as a preferred highfidelity mutant (SacI-HF) where Q117H was a carry over mutation fromtemplate with no affect on activity PvuII Target 47 charged residues andmutate to Ala. No preferred mutants obtained Target 19 hydroxylatedresidues—Ser/Thr and Tyr. Select T46A for further improvement Saturationmutagenesis results in a preferred mutant T46G, T46H, T46K, T46Y.PvuII(T46G) was preferentially selected as a preferred high fidelitymutant (PvuII-HF) MfeI Target 60 charged residues and mutate to Ala. Nopreferred mutants obtained Target 26 hydroxylated residues and mutate toAla except for Tyr which was changed to Phe. Target 38 residues (Cys,Phe, Met, Asn, Gln, Trp) and mutate to Ala Identify Mfe (Q13A/F35Y) as apreferred high fidelity mutant (MfeI-HF) where F35Y is carried from thetemplate HindIII Target 88 charged residues and mutate to Ala. Nopreferred mutants obtained Target 103 residues (Cys Met Asn, Gln, SerThr Trp) and mutate to Ala and Tyr changed to Phe. Identify HindIII(K198A) as a preferred high fidelity mutant (HindIII-HF) SbfI Target 78charged residues mutated to Ala Target 41 residues (Ser Thr) mutated toAla/Tyr to Phe Target 55 residues of Cys, Phe, Met Asn, Gln, Trp to AlaSbfI (K251A) was selected as a preferred high fidelity mutant (SbfI-HF)EagI Target 152 residues (Asp, Glu, His, Lys, Arg, Ser, thr, Asn, andGln changed to Ala and Tyr changed to Phe). EagI H43A was selected as apreferred high fidelity mutant (EagIHF) EcoRV Target 162 residues (Cys,Asp, Glu, Phe, his, Lys, Met, Asn, Gln, Arg, Ser, Thr, to Ala and Trp toPhe) EcoRV (D19A/E27A) was selected as a preferred high fidelity mutant(EcoRV-HF) AvrII Target 210 residues (Cys, Asp, Glu, Phe, his, Lys, Met,Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) AvrII (Y104F) wasselected as a preferred high fidelity mutant (AvrII-HF) BstXI Target 237residues (Cys, Asp, Glu, Phe, his, Lys, Met, Asn, Gln, Arg, Ser, Thr, toAla and Trp to Phe) BstXI (N65A) was selected as a preferred highfidelity mutant (BstXI-HF) PciI Target 151 residues (Cys, Asp, Glu, Phe,his, Lys, Met, Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) PciI(E78A/S133A) was selected as a preferred high fidelity mutant. (PciI-HF)This was spontaneous and not one of the 151 separate mutations HpaITarget 156 residues (Cys, Asp, Glu, Phe, his, Lys, Met, Asn, Gln, Arg,Ser, Thr, to Ala and Trp to Phe) HpaI (E56A) was selected as a preferredhigh fidelity mutant (HpaI-HF) AgeI Target 149 residues (Cys, Asp, Glu,Phe, his, Lys, Met, Asn, Gln, Arg, Ser, Thr, to Ala and Trp to Phe) AgeI(R139A) was selected as a preferred high fidelity mutant (AgeI-HF) BsmBITarget 358 residues (Cys, Asp, Glu, Phe, his, Lys, Met, Asn, Gln, Arg,Ser, Thr, to Ala and Trp to Phe) BsmBI(N185Y/R232A) was selected as apreferred high fidelity mutant (BsmBI (HF) BspQI Target 122 residues(Arg, Lys, His, Glu, Asp, Gln, Asn, Cys) Replace R at position 279 withPhe, Pro, Tyr, Glu, Asp or Leu. Preferred mutations were R388F andK279P. Created a double mutant BspQI(K279P/R388F) as preferred highfidelity mutant (BspQI-HF) SapI Find K273 and R380 in SapI correspondingto R388 and K279 in BspQI. SapI (K273P/R380F) was selected as apreferred high fidelity mutant (SapI-HF) KpnI Target all residues (Asp,Glu, Arg, Lys, His, Ser, Thr, Tyr, Asn, Gln, Phe, Trp, Cys, Met) to Ala.More mutation was done on site D16 and D148. A combined D16N/E132A/D148Ewas selected as a preferred high fidelity mutant (KpnI-HF). BsaI Find 11amino acids corresponding to the site in BsmBI. BsaI (Y231F) wasselected as a preferred high fidelity mutant (BsaI-HF).

The method follows from the realization that amino acids responsible forcognate activity and star activity are different. The engineering ofhigh fidelity restriction endonucleases described herein demonstratesthat cognate activity and star activity can be separated and there aredifferent critical amino acid residues that affect these differentactivities. The locations of amino acids that are here found to affectstar activity are not necessarily found within the active site of theprotein. The cleavage properties of any restriction endonuclease hasbeen determined here for the first time by developing a criterion ofsuccess in the form of determining a FI (see also Wei, et al. NucleicAcid Res., 36, 9, e50 (2008)) and an overall fidelity index improvementfactor.

An “overall fidelity index improvement factor” refers to the highest FIfor a mutant with maximum cleavage activity divided by the highest FI ofthe corresponding WT endonuclease with maximum cleavage activity withina selected set of buffers. The selected set may be of any size greaterthan one but practically will contain less than 10 different buffers andmore preferably contains 4 buffers. The set may also include less than 4buffers. The overall FI improvement factor of at least two shouldpreferably be applicable for any mutant restriction endonuclease in theclaimed invention additionally but not exclusively to the set of buffersconsisting of NEB1, NEB2, NEB3 and NEB4.

A “similar cleavage activity” can be measured by reacting the sameamount of enzyme with the same amount and type of substrate under thesame conditions and visually comparing the cleavage profiles on a gelafter electrophoresis such that the amount of cleavage product appearsto be the same within a standard margin of error and wherein thequantitative similarity is more than 10%.

“Artificial” refers to “man-made”.

“Standard conditions” refers to an overall FI improvement factorcalculated from results obtained in NEB1-4 buffers.

The general method described herein has been exemplified with 27restriction endonucleases: AgeI, AvrII, BamHI, BsaI, BsmBI, BspQI,BstXI, EagI, EcoRI, EcoRV, HindIII, HpaI, KpnI, MfeI, NcoI, NheI, NotI,PciI, PstI, PvuII, SacI, SalI, SapI, SbfI, ScaI, SphI and SspIrestriction endonucleases. However, as mentioned above, the method isexpected to be effective for the engineering of any restrictionendonuclease that has significant star activity.

Embodiments of the method utilize a general approach to create mutantrestriction endonucleases with reduced star activity. For certainenzymes, it has proven useful to mutate charged residues that aredetermined to be conserved between two isoschizomers. In general,however, the method involves a first step of identifying all the chargedand polar residues in a protein sequence for the endonuclease. Forexample, charged amino acids and polar residues include the acidicresidues Glu and Asp, the basic residues His, Lys and Arg, the amideresidues Asn and Gln, the aromatic residues Phe, Tyr and Trp and thenucleophilic residue Cys. Individual residues are targeted and mutatedto an Ala and the products of these targeted mutations are screened forthe desired properties of increased fidelity. If none of the mutantsobtained provide a satisfactory result, the next step is to targetmutations to all the hydroxylated amino acids, namely, Ser, Thr and Tyr,the preferred mutation being Ser and Thr to Ala and Tyr to Phe. It isalso possible to target mutations to both classes of residues at onetime. The mutation to Ala may be substituted by mutations to Val, Leu orIle.

After these analyses, if one or more of the preferred mutants generatedin the above steps still have substandard performance under the selectedtests, these mutants can be selected and mutated again to each of theadditional possible 18 amino acids. This is called saturationmutagenesis. Saturation mutagenesis provided the preferred high fidelitymutants for EcoRI, BamHI in part and PvuII. Depending on the results ofsaturation mutagenesis, the next step would be to introduce additionalmutations either targeted or random or both into the restrictionendonuclease. SacI-HF includes a random mutation generated fortuitouslyduring inverse PCR. PciI-HF resulted from a random mutation and not fromtargeted mutations. BspQI-HF contains two mutations that were found toact synergistically in enhancing fidelity.

The use of various methods of targeted mutagenesis such as inverse PCRmay involve the introduction of non-target mutations at secondary sitesin the protein. These secondary mutations may fortuitously provide thedesired properties. It is desirable to examine those mutated enzymeswith multiple mutations to establish whether all the mutations arerequired for the observed effect. Q117H in the double mutant had noeffect on activity.

In some cases, a mutation may provide an additional advantage other thanimproved fidelity.

The high fidelity/reduced star activity properties of the mutantsprovided in the Examples were selected according to their function in aset of standard buffers. Other mutations may be preferable if differentbuffer compositions were selected. However, the same methodology forfinding mutants would apply. Table 4 lists mutations which apply to eachrestriction endonuclease and provide an overall FI improvement factor inthe standard buffer.

The engineering of the high fidelity restriction endonucleases toprovide an overall FI improvement factor of at least 2 involves one ormore of the following steps:

1. Assessment of the Star Activity of the WT Restriction Endonuclease

In an embodiment of the invention, the extent of star activity of arestriction endonuclease is tested by means of the following protocol:the endonuclease activity is determined for an appropriate substrateusing a high initial concentration of a stock endonuclease and serialdilutions thereof (for example, two-fold or three-fold dilutions). Theinitial concentration of restriction endonuclease is not important aslong as it is sufficient to permit an observation of star activity in atleast one concentration such that on dilution, the star activity is nolonger detected.

An appropriate substrate contains nucleotide sequences that are cleavedby cognate endonuclease activity and where star activity can beobserved. This substrate may be the vector containing the gene for therestriction endonuclease or a second DNA substrate. Examples ofsubstrates used in Table 2 are pBC4, pXba, T7, lambda, and pBR322.

The concentration of stock restriction endonuclease is initiallyselected so that the star activity can be readily recognized and assayedin WT and mutated restriction endonucleases. Appropriate dilutionbuffers such as NEB diluent A, B or C is selected for performing theserial dilutions according to guidelines in the 2007-08 NEB catalog. Theserially diluted restriction endonuclease is reacted with apredetermined concentration of the appropriate substrate in a totalreaction volume that is determined by the size of the reaction vessel.For example, it is convenient to perform multiple reactions inmicrotiter plates where a 30 μl reaction mixture is an appropriatevolume for each well. Hence, the examples generally utilize 0.6 μg ofsubstrate in 30 μl, which is equivalent to 1 μg of substrate in 50 μl.The amount of substrate in the reaction mixture is not critical, but itis preferred that it be constant between reactions. The cleavagereaction occurs at a predetermined temperature (for example 25° C., 30°C., 37° C., 50° C., 55° C. or 65° C.) for a standard time such as onehour. The cleavage products can be determined by any standard technique,for example, by 0.8% agarose gel electrophoresis to determine thefidelity indices as defined above.

Not all restriction endonucleases have significant star activity asdetermined from their FI. However, if an endonuclease has a highest FIof no more than about 250 and a lowest FI of less than 100, therestriction endonuclease is classified as having significant staractivity. Such endonucleases are selected as a target of enzymeengineering to increase fidelity for a single substrate. In some cases,the restriction endonucleases with both FI over about 500 and FI lessthan about 100 are also engineered for better cleavage activity.

Table 2 below lists the FI of some engineered restriction endonucleasesbefore engineering. All samples were analyzed on 0.8% agarose gel.

TABLE 2 Diluent Temp Enzyme (NEB)*** Substrate* ° C. FI-1** FI-2**FI-3** FI-4** AgeI C pXba 37 16 (1) 8 (1/2) 64 (1/8) 8 (1/2) AvrII B T737 64 (1) 8 (1) 32 (1/4) 32 (1) BamHI A λ 37 4 (1/2) 4 (1) 32 (1) 4(1/2) BsaI B pBC4 50 8 (1/4) 120 (1) 16 (1/4) 32 (1) BsmBI B λ 55 1(1/8) 8 (1/2) 120 (1) 4 (1/4) BspQI B λ 50 2 (1/8) 16 (1) 32 (1) 4 (1/2)BstXI B λ 55 2 (1/2) 2 (1/2) 2 (1/8) 4 (1) EagI B pXba 37 4 (1/4) 8(1/2) 250 (1) 16 (1) EcoRI C λ 37 250 (1/2) 4 (1) 250 (1) 4 (1) EcoRV ApXba 37 32 (1/16) 120 (1/2) 1000 (1) 64 (1/4) HindIII B λ 37 32 (1/4)250 (1) 4000 (1/4) 32 (1/2) HpaI A λ 37 32 (1/16) 1 (1/4) 2 (1/8) 16 (1)KpnI A pXba 37 16 (1) 16 (1/4) 8 (1/16) 4 (1/2) MfeI A λ 37 32 (1) 16(1/8) 8 (1/16) 32 (1) NcoI A λ 37 120 (1) 32 (1) 120 (1/4) 32 (1) NheI CpXba 37 32 (1) 120 (1/4) 120 (1/8) 32 (1) NotI C pXba 37 ≧32000 (1/16)64 (1) 500 (1) 32 (1/4) PciI A pXba 37 2000 (1/2) 16 (1/4) 120 (1) 8(1/8) PstI C λ 37 64 (1) 32 (1) 120 (1) 8 (1/2) PvuII A pBR322 37 250(1) 16 (1/4) 8 (1/32) 1/4 (1) SacI A pXba 37 120 (1) 120 (1/2) 120(1/32) 32 (1/2) SalI A λ (H3) 37 8 (1/500) 1 (1/16) 32 (1) 1 (1/120)SapI C λ 37 16 (1/4) 64 (1/2) 32 (1/4) 16 (1) SbfI A λ 37 32 (1) 8 (1/4)8 (1/16) 8 (1/2) ScaI A λ 37 1/16 (1/32) 1/8 (1) 4 (1/2) 1/64 (1/16)SphI B λ 37 64 (1) 32 (1) 64 (1/4) 16 (1/2) SspI C λ 37 64 (1) 16 (1) 32(1/4) 16 (1) *Substrate: λ is lambda phage DNA; λ (H3) isHindIII-digested lambda phage DNA; pXba is pUC19 with XbaI-digestedfragment of Adeno Virus; pBC4: a shorter version of pXba; T7: T7 DNA**FI-1 to FI-4: fidelity index of the enzyme in NEBuffer 1, 2, 3 and 4.The number in parenthesis is a value for relative cleavage activity ofthe mutant restriction endonuclease in a specified buffer in a set ofbuffers compared with the “best” cleavage activity of the same mutantrestriction endonuclease in any of the buffers in the set of buffers.The compositions of NEB buffers follow: NEB1: 10 mM Bis TrisPropane-HCl, 10 mM MgCl₂, 1 mM dithiothreitol (pH 7.0 at 25° C.); NEB2:50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM dithiothreitol (pH 7.9 at25° C.); NEB3: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mMdithiothreitol (pH7.9 at 25° C.); NEB4: 50 mM potassium acetate, 20 mMTris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol (pH7.9 at 25°C.). ***The compositions of NEB diluents follow. (Using diluents in thedilution instead of water will keep the glycerol concentration in thereaction as a constant.) Diluent A: 50 mM KCl, 10 mM Tris-HCl, 0.1 mMEDTA, 1 mM dithiothreitol, 200 mg/ml BSA. 50% glycerol (pH7.4 at 25°C.); Diluent B: 300 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mMdithiothreitol, 500 mg/ml BSA, 50% glycerol (pH7.4 at 25° C.); DiluentC: 250 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.15%Triton X-100, 200 mg/ml BSA, 50% glycerol (pH 7.4 at 25° C.).

2. Construction of High Expression Host Cell Strains

It is convenient if a host cell is capable of over-expressing the mutantrestriction endonuclease for which reduced star activity is sought. Ifthe restriction enzyme is highly expressed in E. coli, the star activitycan be readily detected in the crude extract, which simplifies thescreening for the high fidelity restriction endonuclease. However, themutated restriction endonuclease can be expressed in any host cellproviding that the host cell is protected in some way from toxicityarising from enzyme cleavage.

This might include: the presence of a methylase; production in acompartment of the cell which provides a barrier to access to the genome(such as an inclusion body or the periplasm); in vitro synthesis;production in an emulsion (see U.S. patent application Ser. No.12/035,872) absence of cleavage sites in the host genome; manufacture ofthe enzyme in component parts subject to intein mediated ligation (seeU.S. Pat. No. 6,849,428), etc.

Over-expression of the mutated restriction endonucleases for purposes ofproduction can be achieved using standard techniques of cloning, forexample, use of an E. coli host, insertion of the endonuclease into apUC19-derived expression vector, which is a high copy, and use of arelatively small plasmid that is capable of constant expression ofrecombinant protein. The vector may preferably contain a suitablepromoter such as the lac promoter and a multicopy insertion site placedadjacent to the promoter. Alternatively, a promoter can be selected thatrequires IPTG induction of gene expression. If the activity in the crudeextract is not sufficient, a column purification step for therestriction endonuclease in crude extract may be performed.

3. Mutagenesis of Restriction Endonuclease

DNA encoding each charged or polar group in the restriction endonucleasemay be individually targeted and the mutated DNA cloned and prepared fortesting. Multiple mutations may be introduced into individualrestriction endonuclease genes. Targeted mutagenesis of restrictionendonucleases may be achieved by any method known in the art. Aconvenient method used here is inverse PCR. In this approach, a pair ofcomplementary primers that contains the targeted codon plus a pluralityof nucleotides (for Example 18 nt) on both the 5′ and 3′ side of thecodon is synthesized. The selection of suitable primers can be readilyachieved by reviewing the gene sequence of the endonuclease of interestaround the amino acid residue of interest. Access to gene sequences isprovided through REBASE and GenBank. The template for PCR is a plasmidcontaining the restriction endonuclease gene. The polymerase ispreferably a high fidelity polymerase such as Vent® or Deep Vent™ DNApolymerase. By varying the annealing temperature and Mg²⁺ concentration,successful introduction of most mutations can be achieved. The PCRamplification product is then purified and preferably digested by DpnI.In an embodiment of the invention, the digested product was transformedinto competent host cells (for example, E. coli), which have beenpre-modified with a corresponding methylase. Colonies from each mutantwere picked and grown under similar conditions to those in which the WTis grown (for example, using similar growth medium, drug selection, andtemperature). The resulting restriction endonucleases were screened forreduced star activity.

4. Screening for Mutant Restriction Endonucleases with Reduced StarActivity

Conditions such as buffer composition, temperature and diluent should bedefined for determining star activity in a mutant restrictionendonuclease. Tables 2 and 3 show the FI of recombinant endonucleasesbefore and after mutation in four different buffers using threedifferent diluents at 37° C. Accordingly, it is possible to determinewhich mutants have an overall desirable improved fidelity index factorof at least 2, more than 10, at least 50 or more than 500 and to selectenzymes as preferred high fidelity mutants.

In an embodiment of the invention, the mutant restriction endonucleaseswere screened for activity in normal buffer conditions (no more than 5%glycerol) first. For those mutants with at least about 10% of activityof WT restriction endonuclease, activity was also determined in staractivity promotion conditions that promoted star activity, for example,high glycerol concentration and optionally high pH. Preferably, themutant with the least star activity but with acceptable cognate activityin normal buffers is selected. Plasmid can then be extracted andsequenced for the confirmation of the mutant. In some cases, the staractivity is not easily measured, even with high glycerol and high pHconditions. Instead, the activity in different buffers is measured andcompared, and the one with the highest cleavage activity ratio in NEB4compared with NEB3 can be tested further for star activity improvement.

5. Saturation Mutagenesis on One Single Residue

As described in the previous section, the first step is to mutate atarget amino acid in the restriction endonuclease to Ala. If the resultsare not satisfactory, saturation mutagenesis is performed. This ispreferably performed by one of two methods. One method is to change theintended codon into NNN. After mutagenesis, multiple colonies areassayed under normal conditions and under conditions that promote staractivity. Alternatively, a different codon can be selected formutagenesis of each of the targeted amino acids for example: Ala: GCT;Cys: TGC; Asp: GAC; Glu: GAA; His: CAC; Ile: ATC; Lys: AAA; Leu: CTG;Met: ATG; Asn:

AAC; Pro: CCG; Gln: CAG; Arg: CGT; Ser: TCC; Thr: ACC; Val: GTT; Trp:TGG and Tyr: TAC

6. Combination

More than one mutation can be introduced into the restrictionendonuclease gene if a single mutation does not sufficiently reduce thestar activity. Mutation combination and saturation mutagenesis can beperformed in any order.

7. Mutant Purification and Assessment of the Improvement

The high fidelity mutants may be purified in a variety of ways includinguse of different chromatography columns. For normal quality assessment,one FPLC heparin column is enough to eliminate the DNA and non-specificnucleases from the preparation. Multiple columns including ion exchange,hydrophobic, size exclusion and affinity columns can be used for furtherpurification.

Purified high fidelity restriction endonucleases are measured for FI infour NEB buffers and compared with the FIs of the WT restrictionendonuclease. The ratio of FI for the high fidelity restrictionendonuclease in its optimal buffer to that of WT is the overallimprovement factor.

TABLE 3 FI* for exemplified restriction endonucleases Diluent TempEnzyme (NEB) Substrate* ° C. FI-1** FI-2** FI-3** FI-4** AgeI- C pXba 37≧500 (1) ≧250 (1/2) ≧16 (1/16) ≧250 (1) HF AvrII- B T7 37 500 (1) ≧500(1/2) ≧16 (1/64) ≧1000 (1) HF BamHI- A λ 37 ≧4000 (1) ≧4000 (1) ≧250(1/16) ≧4000 (1) HF BsaI B pBC4 50 ≧4000 (1/2) ≧8000 (1) 120 (1) ≧8000(1) BsmBI B λ 55 2 (1) ≧500 (1) ≧64 (1/8) ≧500 (1) BspQI- A pUC19 50≧1000 (1/4) ≧1000 (1/4) ≧64 (1/64) ≧4000 (1) HF BstXI- A λ 55 ≧120 (1/2)≧250 (1) ≧16 (1/16) ≧250 (1) HF EagI- C pXba 37 250 (1/2) 250 (1) 250(1/2) 500 (1) HF EcoRI- C λ 37 2000 (1/8) 4000 (1/4) 250 (1/250) 16000(1) HF EcoRV- A pXba 37 ≧16000 (1/4) ≧64000 (1) ≧32000 (1/2) ≧64000 (1)HF HindIII- B λ 37 ≧16000 (1/4) ≧64000 (1) ≧16000 (1/4) ≧32000 (1/2) HFHpaI- A λ 37 ≧32 (1/32) ≧2000 (1) 2 (1/8) ≧2000 (1/2) HF KpnI- A pXba 37≧4000 (1) ≧1000 (1/4) ≧64 (1/64) ≧4000 (1) HF MfeI-HF A λ 37 ≧1000 (1)≧250 (1/4) ≧16 (1/64) ≧500 (1/2) NcoI- A λ 37 ≧4000 (1/4) ≧4000 (1/4)≧1000 (1/16) ≧64000 (1) HF NheI- C pXba 37 ≧128000 (1) ≧4000 (1/32) ≧32(1/2000) ≧32000 (1/2) HF NotI-HF C pXba 37 ≧8000 (1/16) ≧128000 (1)≧4000 (1/64) ≧64000 (1/2) PciI-HF A pXba 37 NC ≧2000 (1) ≧2000 (1) ≧1000(1) PstI-HF C λ 37 1000 (1/8) 4000 (1/2) 4000 (1/4) 4000 (1) PvuII- ApBR322 37 ≧250 (1/120) ≧2000 (1/16) ≧250 (1/120) 500 (1) HF SacI- A pXba37 ≧32000 (1) ≧16000 (1/2) ≧500 (1/64) ≧32000 (1) HF SalI-HF A λ (H3) 37≧8000 (1/8) ≧64000 (1) ≧4000 (1/16) ≧32000 (1/2) SbfI-HF C λ 37 1000 (1)120 (1/2) 8 (1/32) 250 (1) ScaI- A λ 37 4000 (1/8) 1000 (1) 2000 (1/32)1000 (1) HF SphI- B λ 37 4000 (1/8) 2000 (1/16) 250 (1/250) 8000 (1) HFSspI- C λ 37 ≧4000 (1/2) 120 (1/2) ≧32 (1/128) 500 (1) HF *The FI is aratio of the highest concentration that does not show star activity tothe lowest concentration that completes digestion of the substrate.**The number in parenthesis is a value for relative cleavage activity ofthe mutant restriction endonuclease in a specified buffer in a set ofbuffers compared with the greatest cleavage activity of the same mutantrestriction endonuclease in any of the buffers in the set of buffers.

TABLE 4 Mutations providing restriction endonucleases with high fidelityRestriction Endonuclease Examples of mutants with overall improved FIfactor ≧ 2 AgeI R139A; S201A* AvrII Y104F; M29A; E96A; K106A; S127A;F142A BamHI E163A/E167T; K30A; E86A; E86P; K87A; K87E; K87V; K87N;P144A; Y165F; E167A; E167R; E167K; E167L; E167I K30A/E86A; E86A/K106A;K30A/E86A/K106A; K30A/K87A; E86P/K87E; E86A/Y165F; K30A/E167A;E163S/E170T/P173A; E163S/E170T/P173A; E86P/K87T/K88N/E163S/E170T/P173A;E86P/K87R/K88G/E163S/E170T/P173A;E86P/K87P/K88R/E163S/E170T/P173A/E211K; E86P/K87T/K88R/ E163S/E170T/P173A/N158S;E86P/K87S/K88P/ E163S/E170T/P173A; E86P/K87G/K88S/E163S/E170T/P173A;E86P/K87R/K88Q/E163S/E170T/P173A; E86P/K87W/K88V; E86P/P173A BsaI Y231FBsmBI N185Y/R232A; H230A; D231A; R232A; BspQI K279P/R388F; K279A; K279F;K279P; K279Y; K279E; K279D R388A; R388F; R388Y; R388L; K279P/R388F;K279A/R388A; D244A BstXI N65A; Y57F; E75A; N76A; K199A; EagI H43A EcoRIK62A; K62S; K62L; R9A; K15A; R123A; K130A; R131A; R183A; S2Y; D135A;R187A; K62E EcoRV D19A; E27A; D19A/E27A HindIII S188P/E190A; K198A HpaIY29F; E56A KpnI D148E; D16N/R119A/D148E; D2A/D16N/D148E;D16N/E134A/D148E; D16N/E132A/D148E MfeI Y173F; Q13A/F35Y NcoI D56A;H143A; E166A; R212A; D268A; A2T/R31A NheI E77A NotI K176A; R177A; R253A;K150A PciI E78A/S133A PstI E204G; K228A; K228A/A289V; D91A PvuII T46A;T46H; T46K; T46Y; T46G SacI Q117H/R154A/L284P; Q117H/R200A SalI R82A;K93A; K101A; R107A SapI K273P; R380A; K273P/R380A SbfI K251A ScaI R18A;R112A; E119A; H193A; S201F; H193A/S201F SphI D91A; D139A; D164A; K100ASspI H65A; K74A; E78A; E85A; E89A; K109A; E118A; R177A; K197A; Y98F Themutations for each enzyme are separated by a semicolon.

All references cited above and below, as well as U.S. Ser. No.12/172,963 filed Jul. 14, 2008 and U.S. provisional application Ser. No.60/959,203, are incorporated by reference.

EXAMPLES

Where amino acids are referred to by a single letter code, this isintended to be standard nomenclature. The key to the code is providedfor example in the NEB catalog 2007/2008 on page 280.

Plasmids used for cloning and as substrates have sequences as follows:

pLaczz2 (SEQ ID NO:102), pSyx20-lacIq (SEQ ID NO:105), pBC4 (SEQ IDNO:103), pXba (SEQ ID. NO:104) and pAGR3 (SEQ ID NO:106). pACYC isdescribed in GenBank XO 6403, T7 in GenBank NC001604, pUC18 in GenBankL09136, and pRRS in Skoglund et al. Gene, 88:1-5 (1990. pSX33 wasconstructed by inserting lad gene into pLG339 at EcoRI site. pLG339 isdescribed in Stoker, et al. Gene 19, 335-341 (1982).

All buffers identified as NEB buffers used herein are obtainable fromNew England Biolabs, Inc. (NEB), Ipswich, Mass.

Example 1 Engineering of High Fidelity NheI 1. Expression of NheI

NheI was expressed in E. coli transformed with pACYC-NheIM, andplaczz1-NheIR. placzz1 is a pUC19 derivative plasmid. The cell was grownat 30° C. for overnight in the LB with Amp and Cam.

2. Mutagenesis of NheI

All 92 charged residues in NheI were mutated to Ala as the followingresidues: 5, 6, 7, 14, 17, 19, 22, 25, 28, 31, 38, 39, 42, 47, 49, 52,56, 58, 59, 60, 64, 74, 75, 76, 77, 80, 91, 93, 104, 105, 110, 112, 116,117, 123, 126, 130, 131, 133, 135, 137, 147, 149, 152, 159, 160, 165,167, 170, 171, 174, 179, 183, 195, 202, 205, 207, 209, 210, 211, 214,216, 218, 221, 225, 231, 241, 243, 244, 250, 252, 256, 257, 259, 264,266, 267, 281, 285, 287, 288, 289, 291, 297, 300, 307, 313, 315, 318,321, 324, 325.

The numbers above correspond to amino acid positions in the NheI proteinsequence (SEQ ID NO:89).

The methods were the same as disclosed in the examples of parentapplication, U.S. Ser. No. 12/172,963, filed Jul. 14, 2008, usinginverse PCR followed by DpnI digestion. The treated product was thentransformed into E. coli (pACYC-NheIM).

3. Selection of NheI-HF

Selection of NheI-HF was performed according to the examples asdisclosed in the parent application, U.S. Ser. No. 12/172,963 filed Jul.14, 2008. The standard and star activity assays contained pBR322 as asubstrate in NEB4 buffer and 5% glycerol and 39% glycerol, respectively.Only one mutation was found to be significant in improving the NheI.This was E77A. NheI(E77A) was selected as the NheI-HF.

4. Comparison of NheI-HF and WT NheI

The FIs of NheI-HF and WT NheI were determined separately on pXba, aplasmid substrate containing the XbaI digested piece from Adeno virus ineach of NEB1-4 buffers. The comparison is shown in FIG. 2A-B, and theresult is listed in Table 14 (below).

TABLE 14 Comparison of NheI-HF and WT NheI NheI-HF WT NheI FidelityFidelity Improvement Buffer Activity Index Activity Index Factor NEB1100% ≧128000 100% 32 ≧4000 NEB2  3% ≧4000  25% 120 ≧32 NEB3 0.05%  ≧3212.5%  120 ≧0.25 NEB4  50% ≧32000 100% 32 ≧1000 NheI-HF showed optimalactivity in NEB1 buffer where its FI is ≧128,000. WT NheI has maximumactivity in NEB1 and NEB4 buffers, where its best FI is 32. The overallFI improvement factor is ≧128,000/32 = ≧4000.

What is claimed is:
 1. A composition, comprising: a restrictionendonuclease having at least one artificially introduced mutation and anoverall fidelity index (FI) improvement factor of at least 2, therestriction endonuclease being capable of cleaving a substrate with atleast a similar cleavage activity to that of the restrictionendonuclease absent the artificially introduced mutation, in apredetermined buffer, wherein the artificially introduced mutation isthe product of at least one of a targeted mutation, saturationmutagenesis, or a mutation introduced through a PCR amplificationprocedure.
 2. A composition, according to claim 1, wherein at least oneof the artificially introduced mutations is a replacement of a naturallyoccurring residue with an oppositely charged residue at a target site inthe restriction endonuclease.
 3. A composition, according to claim 1,wherein at least one of the artificially introduced mutations is areplacement of a naturally occurring residue with a residue selectedfrom a Phenylalanine and an Alanine at a target site in the restrictionendonuclease.
 4. A composition, according to claim 1, wherein therestriction enzyme absent the at least one artificially introducedmutation is selected from the group consisting of: BamHI, EcoRI, Scal,SalI, SphI, PstI, NcoI, NheI, SspI, NotI, SacI, PvuII, MfeI, HindIII,SbfI, EagI, EcoRV, AvrII, BstXI, PciI, HpaI, Agel, BsmBI, BspQI, SapI,KpnI and BsaI.
 5. A composition, according to claim 1, wherein therestriction endonuclease is a variant NheI having reduced star activity,wherein the variant contains a mutation corresponding to position 77 inSEQ ID NO:89.
 6. A composition according to claim 5, wherein themutation is E77A.
 7. A DNA molecule encoding the composition of claim 1.8. A vector containing the DNA of claim
 7. 9. A host cell containing aDNA for expressing the composition of claim
 1. 10. A method, comprising:a) identifying which amino acid residues in an amino acid sequence of arestriction endonuclease having star activity are charged amino acids;b) mutating one or more codons encoding one or more of the chargedresidues in a gene sequence encoding the restriction endonuclease; c)generating a library of gene sequences having one or more differentcodon mutations in different charged residues; d) obtaining a set ofproteins expressed by the mutated gene sequences; and e) determining anFI in a predetermined buffer and a cleavage activity for each protein.11. A method according to claim 10, further comprising: determining anoverall FI improvement factor for proteins belonging to the set ofproteins in a defined set of buffers.
 12. A method according to claim10, wherein the defined set of buffers is comprised of NEB1, NEB2, NEB3and NEB4 buffers.
 13. A method according to claim 10, furthercomprising: mutating codons encoding hydroxylated amino acids in a sameor subsequent step to that of mutating codons for the charged aminoacids.
 14. A method according to claim 10, further comprising: mutatingcodons encoding amide-containing amino acids in a same or subsequentstep to that of mutating the charged amino acids.
 15. A method accordingto claims 10, wherein the codons are mutated to an Alanine except forTyrosine which is mutated to a Phenylalanine.
 16. A method according toclaim 13, wherein the codons are mutated to an Alanine except forTyrosine which is mutated to a Phenylalanine.
 17. A method according toclaim 14, wherein the codons are mutated to an Alanine except forTyrosine which is mutated to a Phenylalanine.
 18. A method according toclaim 12, further comprising: improving the overall FI improvementfactor using saturation mutagenesis of one or more of the mutatedcodons.